The usefulness of semi‐solid medium in the isolation of highly virulent Leptospira strains from wild rats in an urban area of Fukuoka,Japan

Leptospirosis is a worldwide zoonosis. The importance of urban leptospirosis is recognized in Japan: urban rats carry pathogenic leptospires and people acquire these pathogens through contact with surface water or soil contaminated by the urine of the infected animals. To determine the current Leptospira carriage rate in urban rats, 29 wild rats were trapped in the central area of Fukuoka and strains isolated from their kidneys and urine analyzed. When semi‐solid Korthof's medium containing 0.1% agar was used for isolation, 72.2% and 30.8% of the kidney and urine cultures, respectively, were found to be Leptospira‐positive. The isolates belonged to Leptospira interrogans, and were classified into two groups (serogroups Pomona and Icterohaemorrhagiae) based on the results of gyrB sequence analysis and microscopic agglutination testing (MAT). Strains belonging to serogroup Icterohemorrhagiae grew well in liquid medium. On the other hand, serogroup Pomona isolates multiplied very little in liquid medium, but did grow in a semi‐solid medium. Although strains belonging to serogroup Pomona have not been recognized as native to Japan, this strain may be widely distributed in urban rats. Representative strains from each group were found to be highly pathogenic to hamsters. Our findings should serve as a warning that it is still possible to become infected with leptospires from wild rats living in inner cities of Japan. Furthermore, the use of semi‐solid medium for culture will improve the isolation rate of leptospires from the kidneys of wild rats.     

Development of Standardized Insulin Treatment Protocols for Spontaneous Rodent Models of Type 1 Diabetes

Standardized protocols for maintaining near-normal glycemic levels in diabetic rodent models for testing therapeutic agents to treat disease are unavailable. We developed protocols for 2 common models of spontaneous type 1 diabetes, the BioBreeding diabetes-prone (BBDP) rat and nonobese diabetic (NOD) mouse. Insulin formulation, dose level, timing of dose administration, and delivery method were examined and adjusted so that glycemic levels remained within a normal range and fluctuation throughout feeding and resting cycles was minimized. Protamine zinc formulations provided the longest activity, regardless of the source of insulin. Glycemic control with few fluctuations was achieved in diabetic BBDP rats through twice-daily administration of protamine zinc insulin, and results were similar regardless of whether BBDP rats were acutely or chronically diabetic at initiation of treatment. In contrast, glycemic control could not be attained in NOD mice through administration of insulin twice daily. However, glycemic control was achieved in mice through daily administration of 0.25 U insulin through osmotic pumps. Whereas twice-daily injections of protamine zinc insulin provided glycemic control with only minor fluctuations in BBDP rats, mice required continuous delivery of insulin to prevent wide glycemic excursions. Use of these standard protocols likely will aid in the testing of agents to prevent or reverse diabetes.Abbreviations: BBDP, BioBreeding diabetes-prone; BBDR, BioBreeding diabetes-resistant; NOD, nonobese diabetic; PZI, protamine zinc insulin; T1D, type 1 diabetes; VAF, viral-antibody–free; ZT, Zeitgeber timeClinical trials to prevent or reverse type 1 diabetes (T1D) are predicated on preclinical study data obtained from animal models of the disease to determine agents that exhibit efficacy and translational potential. However, according to findings published over the past several years (summarized in references 2, 17, and 31), not all preclinical T1D studies are created equal. Without a standardized screening process, the hundreds of candidate therapeutic agents in development cannot be evaluated critically for translational potential. One parameter that varies considerably from report to report in T1D reversal studies is the insulin treatment provided to diabetic NOD mice. To address the need for standardized preclinical screening of new therapeutics, the National Institute for Diabetes and Digestive and Kidney Diseases has developed the Type 1 Diabetes Preclinical Testing Program.^2,35 Under this program, a central contract testing facility (Biomedical Research Models) bridged the gap between discovery of potential therapeutics and clinical testing for efficacy in prevention or reversal of T1D. Using 2 of the best characterized models of T1D, the BioBreeding diabetes-prone (BBDP) rat and the nonobese diabetic (NOD) mouse, we sought to develop standardized protocols for the treatment of diabetes with insulin to provide the best glycemic control throughout the fed and nonfed states. We began by housing these models in a viral-antibody–free (VAF) barrier facility, we created study designs approved by a scientific advisory board consisting of leaders in the field, and we performed studies by using standard operation procedures.The standard of care in patients with T1D is to attempt to maintain near-normal glucose levels, by providing exogenous insulin therapy several times daily via injection or pump after rigorous monitoring of glycemic levels and by appropriately coordinating insulin dosing with food intake. Current blood glucose control in diabetic rodent models focuses on maintaining the diabetic animal in a state of moderate hyperglycemia, with normal weight gain in the absence of severe ketonuria. This state is achieved by once-daily injections of titrated insulin doses or by implantation of continuous release insulin pellets;³⁸ however, insulin types and methods can vary widely between institutions and laboratories, yielding a wide range of glycemic control. Therefore there is marked difference between the stringent glycemic control targeted by humans with diabetes as compared with the relatively loose glycemic control afforded to rodents with diabetes. Despite the many physiologic differences between humans and rodents, glycemic control potentially can be addressed by making insulin treatment in rodents more comparable in terms of glycemic control to what is achieved currently in humans, especially given that patients with T1D will continue to administer insulin during treatment with therapeutic agents (for example, antiCD3).¹¹ The lessening of the frequency, duration, and severity of hyperglycemic events is anticipated to provide the best chance for β cells to rest (function properly) while interventions are tested.²¹ Ideally, for these studies, animals should receive sufficient insulin to maintain glycemic levels close to the normal range in control nondiabetic animals.For these studies, we focused on the 2 most widely used spontaneous rodent models of T1D: the BioBreeding diabetes-prone (BBDP) rat and the nonobese diabetic (NOD) mouse.^1,12 The BBDP strain originated from a colony of outbred Wistar rats that developed spontaneous diabetes at the BioBreeding Laboratories in the 1970s. In the 1980s, the strain was acquired by the University of Massachusetts Medical School. During inbreeding, the BioBreeding diabetes- resistant (BBDR) control strain was established. Both strains are maintained at our facility and represent the most fully inbred (more than 110 generations) and characterized colonies available. BBDP rats develop T1D at 50 to 90 d of age at a frequency of approximately 85% to 90%, with equal frequency in male and female rats; the disease in BBDP rats results from autoimmune insulitis that is mediated primarily by CD4⁺ and CD8⁺ T cells and the development of autoantibodies to islet antigen. This insulitis is similar to that in human patients.¹⁸ Insulin therapy is required shortly after onset of hyperglycemia or death will occur due to ketoacidosis.¹⁹ The Gimap5 mutation in BBDP rats results in a T-cell lymphopenia and is necessary for development of T1D in BBDP rats (along with expression of a MHC class II RT1 B/D^u allele); adoptive transfer of splenocytes or regulatory T cells from BBDR rats before 35 d of age prevents the onset of diabetes in BBDP rats.^9,28,38 Alternatively, depletion of regulatory T cells from BBDR rats (which are nonlymphopenic) induces T1D in that strain.The NOD mouse strain originated from selective inbreeding of the Cataract Shionogi mouse strain and was imported from Japan to The Joslin Diabetes Center in 1984. NOD mice are now the most widely used preclinical model of T1D, in part due to the availability of genetic analysis and manipulation as well as the wide array of reagents available for mechanistic studies. The most commonly cited source for NOD mice is The Jackson Laboratory (Bar Harbor, ME), where female NOD mice develop disease at a frequency of 65% to 100% by 30 wk of age, whereas male NOD mice develop disease at a frequency of 35% to 85% (inbred for more than 83 generations). The incidence can vary from year to year³⁴ and from facility to facility depending on several factors, the most important being housing conditions.^15,26 The incidence of T1D in female NOD mice at our VAF barrier facility has been 65% to 80% over the past 3 y; this frequency can be far lower in nonVAF facilities. Diabetic NOD mice exhibit mild ketoacidosis, which allows them to survive for as long as several weeks after the onset of hyperglycemia without supportive insulin treatment. NOD mice also present with insulin resistance and a distinct stage of insulitis, referred to as peri-insulitis, that is not found in either human T1D or in diabetic BBDP rats.^5,18 Although both NOD mouse and BBDP rat models of T1D have particular advantages and disadvantages, a prudent path of drug development would include the examination of the therapeutic efficacy of novel agents in both models.^2,31To standardize and improve current testing protocols, we developed insulin treatment regimens that maintain blood glucose levels near normal levels throughout day and night activities over prolonged periods, as would be expected to occur in interventional clinical trials. We show here that whereas 2 daily injections of insulin to diabetic BBDP rats were sufficient to achieve our goal, diabetic NOD mice required continuous delivery of insulin through the implantation of osmotic pumps.     

Adipocyte-specific deficiency of angiotensinogen decreases plasma angiotensinogen concentration and systolic blood pressure in mice

Previous studies demonstrated that overexpression of angiotensinogen (AGT) in adipose tissue increased blood pressure. However, the contribution of endogenous AGT in adipocytes to the systemic renin-angiotensin system (RAS) and blood pressure control is undefined. To define a role of adipocyte-derived AGT, mice with loxP sites flanking exon 2 of the AGT gene (Agt(fl/fl)) were bred to transgenic mice expressing Cre recombinase under the control of an adipocyte fatty acid-binding protein 4 promoter (aP2) promoter to generate mice with adipocyte AGT deficiency (Agt(aP2)). AGT mRNA abundance in adipose tissue and AGT secretion from adipocytes were reduced markedly in adipose tissues of Agt(aP2) mice. To determine the contribution of adipocyte-derived AGT to the systemic RAS and blood pressure control, mice were fed normal laboratory diet for 2 or 12 mo. In males and females of each genotype, body weight and fat mass increased with age. However, there was no effect of adipocyte AGT deficiency on body weight, fat mass, or adipocyte size. At 2 and 12 mo of age, mice with deficiency of AGT in adipocytes had reduced plasma concentrations of AGT (by 24-28%) compared with controls. Moreover, mice lacking AGT in adipocytes exhibited reduced systolic blood pressures compared with controls (Agt(fl/fl), 117 ± 2; Agt(aP2), 110 ± 2 mmHg; P < 0.05). These results demonstrate that adipocyte-derived AGT contributes to the systemic RAS and blood pressure control.     

Novel and selective spiroindoline-based inhibitors of Sky kinase

We report the discovery of a novel series of spiroindoline-based inhibitors of Sky kinase that bind in the ATP-binding site and exhibit high levels of kinome selectivity through filling the Ala571-subpocket. These inhibitors exhibit moderate oral bioavailability in the rat due to low absorption across the gut wall.     

The fbpA/sapM double knock out strain of Mycobacterium tuberculosis is highly attenuated and immunogenic in macrophages

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine.     

Mig-6 plays a critical role in the regulation of cholesterol homeostasis and bile Acid synthesis

The disruption of cholesterol homeostasis leads to an increase in cholesterol levels which results in the development of cardiovascular disease. Mitogen Inducible Gene 6 (Mig-6) is an immediate early response gene that can be induced by various mitogens, stresses, and hormones. To identify the metabolic role of Mig-6 in the liver, we conditionally ablated Mig-6 in the liver using the Albumin-Cre mouse model (Alb(cre/+)Mig-6(f/f); Mig-6(d/d)). Mig-6(d/d) mice exhibit hepatomegaly and fatty liver. Serum levels of total, LDL, and HDL cholesterol and hepatic lipid were significantly increased in the Mig-6(d/d) mice. The daily excretion of fecal bile acids was significantly decreased in the Mig-6(d/d) mice. DNA microarray analysis of mRNA isolated from the livers of these mice showed alterations in genes that regulate lipid metabolism, bile acid, and cholesterol synthesis, while the expression of genes that regulate biliary excretion of bile acid and triglyceride synthesis showed no difference in the Mig-6(d/d) mice compared to Mig-6(f/f) controls. These results indicate that Mig-6 plays an important role in cholesterol homeostasis and bile acid synthesis. Mice with liver specific conditional ablation of Mig-6 develop hepatomegaly and increased intrahepatic lipid and provide a novel model system to investigate the genetic and molecular events involved in the regulation of cholesterol homeostasis and bile acid synthesis. Defining the molecular mechanisms by which Mig-6 regulates cholesterol homeostasis will provide new insights into the development of more effective ways for the treatment and prevention of cardiovascular disease.     

Reducing environmental bias when measuring natural selection

Abstract.— Crucial to understanding the process of natural selection is characterizing phenotypic selection. Measures of phenotypic selection can be biased by environmental variation among individuals that causes a spurious correlation between a trait and fitness. One solution is analyzing genotypic data, rather than phenotypic data. Genotypic data, however, are difficult to gather, can be gathered from few species, and typically have low statistical power. Environmental correlations may act through traits other than through fitness itself. A path analytic framework, which includes measures of such traits, may reduce environmental bias in estimates of selection coefficients. We tested the efficacy of path analysis to reduce bias by re-analyzing three experiments where both phenotypic and genotypic data were available. All three consisted of plant species (Impatiens capensis, Arabidopsis thaliana , and Raphanus sativus) grown in experimental plots or the greenhouse. We found that selection coefficients estimated by path analysis using phenotypic data were highly correlated with those based on genotypic data with little systematic bias in estimating the strength of selection. Although not a panacea, using path analysis can substantially reduce environmental biases in estimates of selection coefficients. Such confidence in phenotypic selection estimates is critical for progress in the study of natural selection.